Malaria is responsible for several hundred million cases and over one million deaths per year. A highly effective vaccine that prevents blood stage infection and thereby prevents all disease and transmission would be an ideal tool for eliminating Plasmodium falciparum (Pf), the cause of 99% of malaria mortality. Attenuated Pf sporozoites (PfSPZ) administered by the bite of infected mosquitoes, lead to protection of >90% of human volunteers against experimental Pf challenge that lasts at least 10 months. Sanaria's goal is to develop and commercialize an attenuated PfSPZ vaccine that prevents Pf blood stage infection in >90% of recipients - a vaccine with these characteristics could be used to eliminate Pf from the world. This vaccine has the potential for >$1 billion annual revenues. Sanaria has successfully established robust, reproducible, and consistent manufacture and release of clinical lots of its PfSPZ Vaccine, received FDA approval (IND) for clinical trials, and initiated a Phase 1 clinical trial to assess safety, immunogenicity, and protective efficacy of the PfSPZ Vaccine in May 2009. Additional proof of concept studies in African adults, young children, and infants are planned and will be followed by Phase 2 studies, and then pivotal Phase 3 studies to support licensure. Sanaria's manufacturing process utilizes a master cell bank of cryopreserved P. falciparum-infected erythrocytes as a source of parasites, and Anopheles stephensi mosquitoes to raise the PfSPZ. However, because there is no method for cryopreserving any stage of the Anopheles mosquito life cycle, the mosquitoes used for producing the PfSPZ Vaccine come from a mosquito colony that is continuously maintained. Cryopreservation of the eggs of Anopheles spp. would enable Sanaria to cryobank different strains of An. stephensi, reduce costs associated with strain maintenance, produce a uniform seed stock and provide security against strain loss or genetic drift. Cryobanking of transgenic An. stephensi and other Anopheles spp. would also provide direct benefit to those aiming to use genetically altered or sterile mosquitoes for transmission control. In pilot studies, we have shown that An. stephensi can be cryopreserved in the vapor phase of liquid nitrogen using a novel cryoprotectant additive (CPA) mixture, with successful hatching and larval development following thawing. An invention disclosure describing the details that led to the production of the first successful Anopheles egg cryopreservation has been filed. The hatch rate to date has been 10% compared to non-cryopreserved controls from the same egg batch which is clear indication of proof of concept. This proposal will define and optimize the successful cryopreservation of An. stephensi by a pragmatic and systematic testing of embryos at different stages of development and of cryopreservation methods and additives. Successful outcome will be measured as the development from cryopreserved eggs of An. stephensi that retain full susceptibility to infection with PfSPZ in the salivary glands. Other strains of An. stephensi held by Sanaria, including transgenic strains, will be similarly cryopreserved. PUBLIC HEALTH RELEVANCE: Malaria causes 500 million clinical cases and 1-3 million deaths annually, is responsible for >1% loss of GDP in Africa annually and is a serious concern for travelers and military personnel. Sanaria's goal is to develop and commercialize a >90% protective malaria vaccine for three primary markets with a potential for >$1 billion annual revenues. Success in this project will represent a major breakthrough in the fields of vector and malaria biology, will ensure the ability to conserve the genetic stock of the mosquitoes used by Sanaria's in its vaccine manufacturing process, will move Sanaria towards ensuring consistency of mosquito colonies across different manufacturing sites and will provide a secure cryobank of material from which mosquito production can be initiated periodically and which can be followed in a well-defined stability program.